RUI (MCB - Genetic Mechanisms): Molecular analysis of two interacting components of the conjugation machinery of Bacillus subtilis

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Bacteria have the remarkable ability to acquire new genes in a process known as conjugation, which plays a profound role in bacterial evolution and adaptation to different environments. During conjugation, DNA is transferred from one cell to another through a specialized translocation channel. Many of the molecular mechanisms behind the conjugation process remain a mystery. This project will characterize two critical interacting protein components of the conjugation machinery of the bacterium Bacillus subtilis, providing insight into how they function, interact, and localize. The research will be designed and conducted by 12 highly qualified and diverse undergraduate students. By attaining this independent, inquiry-driven research experience, students are more likely to remain in science and gain employment or entrance to top-tier graduate programs after graduation. This project will also improve science education by enabling a professor from a diverse community college to do research and learn new techniques.The subject of this study is a specialized DNA translocation channel known as a Type IV secretion system (T4SS). While the T4SSs found in several Gram-negative bacteria have been well-studied, much less is known about those of Gram-positive bacteria. This project aims to examine the conjugation machinery of Gram-positive bacteria using the integrative and conjugative element ICEBs1 of B. subtilis as a model system. Prior results indicate that the ICEBs1 T4SS is composed of ConE and ConB, among other proteins. ConE is a peripheral membrane ATPase that likely energizes DNA transfer and/or channel assembly. The bitopic membrane protein ConB interacts with ConE and is required for its association with the membrane. ConB and ConE will be characterized by a variety of methods including mutagenesis, conjugation assays, fluorescence microscopy, and biochemistry. This research will define the conserved sequence motifs and domains of ConB and ConE that are required for conjugation, interaction, enzymatic function, and oligomerization. As these proteins are conserved, the findings will apply to the conjugation machinery of numerous Gram-positive bacteria and provide a deeper understanding of a major mechanism mediating horizontal gene transfer.